Science Behind

THE POWER OF FLOW CYTOMETRY – NOW ALSO FOR HEMOSTASIS

In flow cytometry, single cells are passed through one or more laser beams, where the light scatter (indicating cell size and granularity) and signal intensity from different fluorescently labeled markers bound to the cell can be measured.

Labelling platelets or extracellular vesicles (EVs) - sometimes called microparticles - with antibodies directed against surface membrane glycoproteins and then analyzing the binding by flow cytometry is a simple, rapid, sensitive, reliable and cost-effective technique which is eminently suited to study the expression of platelet surface receptors, both qualitatively as well as quantitatively with wide application in the diagnosis of various inherited and acquired platelet disorders.

The possibility to obtain results from thousands of individual platelets in just a few seconds, and from just a few microliters of blood is also attractive, especially for pediatric patients

Modern flow cytometers and fluorescent antibodies enable several antigens to be measured simultaneously on each cell assayed, together with forward and side light scatter properties that correlate with cell size and internal complexity or granularity.

Emosis goal is to use the power of flow cytometry and apply it to support the hemostasis community, looking at specific markers of platelets or microparticles in specific clinical conditions, e.g. looking at thrombotic or bleeding risk. Flow cytometry can also be used to study and compare the effect of various antiplatelet drugs on the level of platelet activation and to establish any dose-effect relationship of these drugs.

THE CLASSICAL MODEL OF HEMOSTASIS

Hemostasis or haemostasis (from the Greek words haimo, ’blood’, and stasis, ’to stop’) is the set of mechanisms that maintains blood in a fluid state under normal conditions and responds to vessel damage by the rapid formation of a clot. The biochemistry of hemostasis is pretty complex. In an effort to understand it, a “cascade/waterfall” model was developed in the mid-60s and has been widely accepted among the medical and scientific communities ever since. According to this model, inactive clotting factors present in blood, are converted into active enzymes in a step-by-step sequence, where each protease activates the subsequent one in the series by means of proteolytic reactions. This process finally results in the production of large amounts of thrombin and subsequent formation of a fibrin clot. Although applicable to the laboratory coagulation tests, it is clearly inadequate to explain the pathways leading to hemostasis in vivo.

For that reason, in the last decade, Hoffman and Monroe have proposed a new model of homeostasis (A Cell-based Model of Hemostasis, Thromb Haemost 2001; 85: 958–65) where coagulation is regulated by the properties of cell surfaces with specific receptors for the coagulation factors. Their model explains some aspects of hemostasis that the “cascade”, protein-centric model cannot explain.

THE NEWER CELL-BASED HEMOSTASIS MODEL

When compared with the traditional cascade model, the cell-based conceptual model of haemostasis allows a more fundamental understanding of the clinical problems observed in some coagulation disorders by focusing on the central role of specific cell surfaces in controlling and directing the haemostatic process.

In the cell-based model of hemostasis, coagulation takes place in three overlapping stages:

Initiation: Initiation occurs on a tissue-factor bearing cell. If the stimulus is strong enough, factors Xa, IXa and thrombin are formed to successfully initiate the coagulation process.

Propagation: factor complexes “tenase” and “prothrombinase” are formed on the platelet surface and large amounts of thrombin are generated.

This cell-based model explains some aspects of hemostasis that a protein-centric model does not.

PLATELETS AND THEIR KEY ROLE IN HEMOSTASIS

Platelets play a paramount role in the coagulation process, but being technically more demanding to explore in a laboratory, are poorly taken into account today in coagulation tests panels. The primary role of platelets in hemostasis is to support coagulation by the formation of a platelet plug comprised of aggregated platelets, and to provide a catalytic surface for the generation of thrombin for fibrin clot formation

Microparticles (MPs), discovered in 1967 by Peter Wolf as a latelet dust”, are present in the blood of healthy individuals and are increased in various diseases, including cardiovascular disease, diabetes, sepsis and cancer. Microparticles are 0.1–1 micrometer cell-derived vesicles that lack a nucleus or synthetic capacity, may contain cytoskeletal proteins, and expose some quantity of phosphatidylserine on their surfaces. MPs have been proposed to play roles in thrombosis, inflammation and angiogenesis.

EMOSIS – BRINGING FLOW CYTOMETRY TESTS TO HEMOSTASIS LABORATORIES

On the contrary, Emosis tests assess platelets’ behavior in minute details and, in so-doing, provide a more informative, accurate, and quantitative complete overview of the underlying hemostasis abnormality by revealing where there may be a problem and possibly the extent of that problem. In addition, using minimal blood sampling, the tests can be performed in patient populations who, at present, have no access to current tests (e.g. neonates).

Furthermore, Emosis tests would be the first routine cell-based hemostasis tests to be performed either on existing flow cytometers (available in major hospitals, although they are usually very busy with oncology or HIV testing), or even more conveniently on a new generation of benchtop flow cytometers – a technology known for its power, accuracy and precision with the convenience of an easier-to-use profile. Unlike many existing tests, Emosis tests can be performed without specialized cytometrists, are accessible to any typically-equipped hospital laboratory and can be performed quickly and routinely at affordable costs.

Of growing importance is the fact that quicker and more actionable laboratory results can strongly reduce the costs for each hospital and for the health care system, through shorter length of stay (e.g., in expensive ICU) and through lower use of expensive alternative anticoagulant drugs.

Please find below the white paper written by Jean Amiral, the CTO of Emosis : " Smart and Friendly to Use Functional Assay for Confirmation of Heparin-Induced Thrombocytopenia - Combination with Clinical Probability and Immunoassay for the Diagnosis of HIT "